Defective RNA sensing by RIG-I in severe influenza virus infection.

Influenza virus infection causes worldwide seasonal epidemics. Although influenza is usually a mild disease, a minority of patients experience very severe fulminating disease courses. Previous studies have demonstrated a role for type I interferon (IFN) in anti-viral responses during influenza. So far, however, IFN regulatory factor (IRF)7 deficiency is the only genetic cause of severe influenza described in humans. In this study we present a patient with severe influenza A virus (IAV) H1N1 infection during the 2009 swine flu pandemic. By whole exome sequencing we identified two variants, p.R71H and p.P885S, located in the caspase activation and recruitment domain (CARD) and RNA binding domains, respectively, of DExD/H-box helicase 58 (DDX58) encoding the RNA sensor retinoic acid inducible gene 1 (RIG-I). These variants significantly impair the signalling activity of RIG-I. Similarly, patient cells demonstrate decreased antiviral responses to RIG-I ligands as well as increased proinflammatory responses to IAV, suggesting dysregulation of the innate immune response with increased immunopathology. We suggest that these RIG-I variants may have contributed to severe influenza in this patient and advocate that RIG-I variants should be sought in future studies of genetic factors influencing single-stranded RNA virus infections.

Sorption behavior of Eu(III) from an aqueous solution onto modified hydroxyapatite: kinetics, modeling and thermodynamics.

Nano-pore hydroxyapatite (HAP) was prepared using physical activation of raw and chemically modified [using Acid; HNO3 (HAPA) or Base; NaOH (HAPB)] bone char (BC) by heating at 900°C for 1 hr to obtain HAP9, HAPA9 and HAPB9, respectively. Investigation the effects of thermal and chemical treatment of prepared nano-hydroxyapatite on elemental analysis, FTIR, scanning electron microscopy, surface area and consequently, the sorption behavior of Eu (III) ions onto the prepared nano-pore hydroxyapatite. Batch adsorption technique was used and the obtained results revealed that the optimum pH = 5.0. The % removal of europium (III) using HAPA9 and HAPB9 reach to 100% within 15 min, while HAP9 after 180 min and the pseudo-second-order was found to be fit to the experimental data. According to Langmuir model, the maximum sorption capacities (qm) were 123.8, 384.9 and 74.2 mg g-1 for HAP9, HAPA9 and HAPB9, respectively. The reaction is spontaneous according to ΔG° value. HCl (0.5 M) was the most efficient desorbing agent for recovery of Eu(III) and regeneration of adsorbents. Finally, nano-pore hydroxyapatite (HAP) was low cost and very effective adsorbent for sorption or recovery of Eu(III) from aqueous solutions and remediation of environmental pollution.

Validation and development of MTH1 inhibitors for treatment of cancer.

BACKGROUND: Previously, we showed cancer cells rely on the MTH1 protein to prevent incorporation of otherwise deadly oxidised nucleotides into DNA and we developed MTH1 inhibitors which selectively kill cancer cells. Recently, several new and potent inhibitors of MTH1 were demonstrated to be non-toxic to cancer cells, challenging the utility of MTH1 inhibition as a target for cancer treatment. MATERIAL AND METHODS: Human cancer cell lines were exposed in vitro to MTH1 inhibitors or depleted of MTH1 by siRNA or shRNA. 8-oxodG was measured by immunostaining and modified comet assay. Thermal Proteome profiling, proteomics, cellular thermal shift assays, kinase and CEREP panel were used for target engagement, mode of action and selectivity investigations of MTH1 inhibitors. Effect of MTH1 inhibition on tumour growth was explored in BRAF V600E-mutated malignant melanoma patient derived xenograft and human colon cancer SW480 and HCT116 xenograft models. RESULTS: Here, we demonstrate that recently described MTH1 inhibitors, which fail to kill cancer cells, also fail to introduce the toxic oxidized nucleotides into DNA. We also describe a new MTH1 inhibitor TH1579, (Karonudib), an analogue of TH588, which is a potent, selective MTH1 inhibitor with good oral availability and demonstrates excellent pharmacokinetic and anti-cancer properties in vivo. CONCLUSION: We demonstrate that in order to kill cancer cells MTH1 inhibitors must also introduce oxidized nucleotides into DNA. Furthermore, we describe TH1579 as a best-in-class MTH1 inhibitor, which we expect to be useful in order to further validate the MTH1 inhibitor concept.

Overexpression of the scaffold WD40 protein WRAP53ß enhances the repair of and cell survival from DNA double-strand breaks.

Altered expression of the multifunctional protein WRAP53ß (WD40 encoding RNA Antisense to p53), which targets repair factors to DNA double-strand breaks and factors involved in telomere elongation to Cajal bodies, is linked to carcinogenesis. While loss of WRAP53ß function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53ß disrupts the formation of and impairs the localization of coilin to Cajal bodies. At the same time, the function of this protein in the repair of DNA double-strand breaks is enhanced. Following irradiation, cells overexpressing WRAP53ß exhibit more rapid clearance of phospho-histone H2AX (γH2AX), and more efficient homologous recombination and non-homologous end-joining, in association with fewer DNA breaks. Moreover, in these cells the ubiquitylation of damaged chromatin, which is known to facilitate the recruitment of repair factors and subsequent repair, is elevated. Knockdown of the ubiquitin ligase involved, ring-finger protein 8 (RNF8), which is recruited to DNA breaks by WRAP53ß, attenuated this effect, suggesting that overexpression of WRAP53ß leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53ß and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53ß may contribute to genomic stability in and survival of cancer cells.

Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells.

The IFNL4 gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis C virus infection. The activity of IFNλ4 has an important causal role in the pathogenesis, but the molecular details are not fully understood. One possible reason for the detrimental effect of IFNλ4 could be a tissue-specific regulation of an unknown subset of genes. To address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with IFNα, IFNλ3 or IFNλ4 and assessed interferon mediated gene regulation using transcriptome sequencing. Our data show a surprisingly similar response to all three subtypes of interferon. We also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. However, the interferon response is robust in both tissues with the majority of the identified genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium.

A conserved sugar bridge connected to the WSXWS motif has an important role for transport of IL-21R to the plasma membrane.

Interleukin-21 (IL-21) is a class I cytokine that belongs to the γc-subfamily of cytokines and regulates immune responses. It signals through a heterodimeric receptor complex composed of the IL-21R1 and γc-receptor chains. A characteristic feature of class I cytokine receptors is the presence of a consensus motif WSXWS (WS motif) in the membrane proximal fibronectin type III domain (FNIII) of these receptors. We recently described the structure of the IL-21R:IL-21 complex and showed that the first tryptophan of the WS motif of IL-21R is mannosylated and involved in formation of a sugar bridge that connects the two FNIII domains of the receptor. Furthermore, a mutation within the WS motif of IL-21R was recently shown to cause a novel kind of primary immunodeficiency syndrome (PID). Here, we report the structure of IL-21R alone, which shows that the sugar bridge forms independently of whether IL-21R binds IL-21 or not, and we furthermore investigate the role of this bridge in the export of IL-21R and γC to the plasma membrane. Thus, we provide a molecular explanation for how mutations in the WS motif may cause PIDs.

Effect of different managerial systems on productive and reproductive traits, blood plasma hormones and serum biochemical constituents of geese.

A flock of 117, 10-month-old Egyptian geese consisting of 90 females and 27 males were utilized in this investigation. Birds were randomly divided into three equal groups, each made up of three replicates of 10 females and 3 males each. The first group was kept under a pasture system (PS) and allowed to swim in water ducts during the daytime (PS) and kept inside the house during the night. The second group of birds were kept in confinement in a house and fed ad libitum on a commercial feed (intensive system (IS)). Birds in the third group (semi-intensive system (SIS)) were released from the house for 6 h a day and given access to the pasture and water ducts. Each group was housed in three pens (replicates) in the SIS. They were given ad libitum access to the commercial feed when in the house. Each pen measured (2 × 3 m2). Natural mating was practiced during the period from November to the end of May. BW of geese under ISS was significantly (P ⩽ 0.05) higher than those under PS and SIS. Egg number, weight and mass of geese in the SIS system were significantly (P ⩽ 0.05) greater than those of geese in the PS and IS systems. Fertility and hatchability percentages were significantly (P ⩽ 0.05) greater in the PS (84.2% and 88.6%) than in the IS (77.5% and 82.8%) and SIS systems (80.7% and 85.5%). Shell weight and thickness were significantly (P ⩽ 0.05) better in the IS and SIS systems than in the PS system. Geese in the PS and SIS systems exhibited significantly higher plasma estradiol-17 and progesterone than those in the IS. Testosterone was significantly higher in IS than in the other systems. Semen quality factor was significantly higher in the PS and SIS systems than in the IS system. Carcass weight was significantly greater in IS and SIS geese than in PS geese, but the PS system resulted in a decreased percentage skin, abdominal fat and liver. Total amount of meat produced per geese was significantly greater in the SIS than in the IS system and greater in the IS than in the PS system.

Radiologically assessed testicular changes in infertile males with varicocele.

The aim of this study was to assess vascular disturbances in the testis of infertile males associated with varicocele. In total, 124 consecutive male subjects were divided into the following groups: healthy fertile controls (n = 10), Gp1 (n = 28); infertile males with subclinical varicocele, Gp2 (n = 26); infertile males with grade I left varicocele, Gp3 (n = 28); infertile males with grade II left varicocele and Gp4 (n = 32); infertile males with grade III left varicocele. They were subjected to colour duplex for pampiniform plexus and scrotal scintigraphy. There was significant decrease in arterial blood velocity, testicular arterial diameters and testicular perfusion especially in high grade varicocele compared with healthy controls. The mean vein diameter demonstrated significant negative correlation with arterial diameter, arterial blood velocity, perfusion index, testicular size and significant positive correlation with perfusion index. The mean testicular size demonstrated significant positive correlation with arterial blood velocity, perfusion index and nonsignificant correlation with arterial diameter. It is concluded that there are significant decreases in testicular volume, testicular perfusion, blood velocity and testicular artery diameter in infertile males with varicocele.

Sorption of lanthanum and erbium from aqueous solution by activated carbon prepared from rice husk.

A biomass agricultural waste material, rice husk (RH) was used for preparation of activated carbon by chemical activation using phosphoric acid. The effect of various factors, e.g. time, pH, initial concentration and temperature of carbon on the adsorption capacity of lanthanum and erbium was quantitatively determined. It was found that the monolayer capacity is 175.4 mg g(-1) for La(III) and 250 mg g(-1) for Er(III). The calculated activation energy of La(III) adsorption on the activated carbon derived from rice husk was equal to 5.84 kJ/mol while it was 3.6 kJ/mol for Er(III), which confirm that the reaction is mainly particle-diffusion-controlled. The kinetics of sorption was described by a model of a pseudo-second-order. External diffusion and intra-particular diffusion were examined. The experimental data show that the external diffusion and intra-particular diffusion are significant in the determination of the sorption rate. Therefore, the developed sorbent is considered as a better replacement technology for removal of La(III) and Er(III) ions from aqueous solution due to its low-cost and good efficiency, fast kinetics, as well as easy to handle and thus no or small amount of secondary sludge is obtained in this application.

Human interferon-lambda3 is a potent member of the type III interferon family.

Type III interferon (IFN) or IFN-lambda is a recently discovered family of IFNs that signal through the same downstream transcription factors as type I IFN but use a separate receptor complex composed of the IL-10R2 and the unique IFN-lambdaR1 receptor chains. We have established a simple and efficient expression system to produce highly pure and active IFN-lambda of the three human IFN-lambda isoforms (IFN-lambda1, -lambda2 and -lambda3) and used this to compare the biological activity of the different IFN-lambda subtypes. Surprisingly, we found IFN-lambda3 to possess the highest specific activity of the human IFN-lambda subtypes, exhibiting a twofold higher activity than IFN-lambda1 and a 16-fold higher activity than IFN-lambda2. Furthermore, in comparison with the commercially available preparations of IFN-lambda1 and -lambda2, we found our IFN-lambda preparation to be superior in activity.

Malignant bone tumors in Kuwait: a 10-year clinicopathological study.

Between 1995 and 2004, 142 malignant bone tumours comprising 76 primary and 66 secondary tumours were identified in the Pathology Department of Al-Sabah Hospital, Kuwait. Pathological fracture was the presenting sign in 35% of the cases. The mean incidence of primary tumours/year was 3.3 cases/million inhabitants. The primary tumours showed a male predilection and 42% occurred below the age of 20 years. The most frequent in the descending order of frequency were Ewing's sarcoma, multiple myeloma, osteosarcoma, chondrosarcoma and non-Hodgkin's lymphoma. The femur was the most common site for secondary tumours; more than half of the tumours with metastases at this site originated in the breast. The high frequency of Ewing's sarcoma is noteworthy and requires further investigation.

Some epidemiological aspects of needle stick injuries among the hospital health care workers: Eastern Province, Saudi Arabia.

This study was conducted to provide some epidemiological aspects of needle stick injuries among health care workers in the eastern province in Saudi Arabia. Data about 282 injuries reported from the hospitals of the Eastern Province of Saudi Arabia were collected through specially designed questionnaire retrospectively from 1995 to 1997. Results showed that 50% of injuries occurred in the first 3 years of employment. Workers in medical and surgical specialties suffered an equal degree of reported exposure. The highest percentage (46.8%) of injuries occurred during syringe related actions. Patients' wards were the major location of incidents (48.5%) followed by ICUs and dialysis units (17.7%), theatres (15.6%) and accidents and emergency departments (13.8%). There was no significant association between injuries and type of shifts. The middle hours of the shifts showed a significant association with injuries. The pattern of injuries was significantly associated with over loaded periods of medical practices. Deficiencies in implementing the standard recommendations for prevention and control of needle stick injuries were noted in the studied hospitals.

Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin.

Coordination between sequential steps in synaptic vesicle endocytosis, including clathrin coat formation, fission, and uncoating, appears to involve proteinprotein interactions. Here, we show that compounds that disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis in a living synapse. Two distinct endocytic intermediates accumulated. Free clathrin-coated vesicles were induced by a peptide-blocking endophilin's SH3 domain and by antibodies to the proline-rich domain (PRD) of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. We suggest that the SH3 domain of endophilin participates in both fission and uncoating and that it may be a key component of a molecular switch that couples the fission reaction to uncoating.

Synthesis of new uracil-5-sulphonamide-p-phenyl derivatives and their effect on Biomphalaria alexandrina snail's nucleoproteins.

In continuation of the previous work (Fathalla, 1992) on the synthesis of some heterocycles containing uracil moiety, we report herein the incorporation of uracil moiety into cyanopyridine thione, thiosemecarbazone, semicarbazone, cyanopyridine, aminocyano pyridine, isoxazoline, pyrazoline, pyrimidine, triazolo pyrimidine, pyran, selena and thiazole derivatives which might modify their biological activities. The biological studies revealed that the chemical compound III f showed high molluscicdal activity than other compounds. The profile of the nucleoprotein extracted from chemically (compound IIIc, e, f and g) treated or UV-irradiated B. alexandrina snails did not show appreciable differences when compared to non-treated (native) snails by using SDS-PAGE, where no obvious qualitative or quantitative differences were observed. Immunization of experimental animals with the nucleoprotein extracted from native, chemically (compound III f & g) treated or physically treated B. alexandrina snails induced significant protection against challenge with normal S. mansoni cercariae, as compared to the non-immunized challenged control. As well as, a decrease in the number of granuloma formation and the size range of granuloma was also observed in immunized animals. It is concluded that, compounds III f and g have a potent molluscicidal activity. They also induced chemical modification comparable to that induced by physical treatment in the snail's nucleoprotein, which could possibly be used in immunization against S. mansoni infection.

Frequency of and prevention measures for needle-stick injuries among hospital healthcare workers in Saudi Arabia.

This study was undertaken to determine the frequency of needle-stick injuries among healthcare workers in hospitals in the Eastern Province of Saudi Arabia from 1995 to 1997, and to gather information about the measures these hospitals are taking to protect their employees against these injuries. Precautionary measures taken to protect healthcare workers from injury and the follow-up after injuries were surveyed. The total overall prevalence rate of needle-stick injuries was significantly lower among healthcare workers in governmental hospitals than among those in private hospitals. The extremely low prevalence rate of needle-stick injuries indicated that there were serious defects in the reporting systems of the hospitals studied. All hospitals should have a mandatory, clear, and unified policy to help reduce the number of needle-stick injuries. Hospitals must adopt a policy to ensure that precautions are taken to reduce healthcare workers' accidental exposures to potentially infected blood.

Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis.

Endophilin/SH3p4 is a protein highly enriched in nerve terminals that binds the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin, two proteins implicated in synaptic vesicle endocytosis. We show here that antibody-mediated disruption of endophilin function in a tonically stimulated synapse leads to a block in the invagination of clathrin-coated pits adjacent to the active zone and therefore to a block of synaptic vesicle recycling. We also show that in a cell-free system, endophilin is not associated with clathrin coats and is a functional partner of dynamin. Our findings suggest that endophilin is part of a biochemical machinery that acts in trans to the clathrin coat from early stages to vesicle fission.

Dissociation between Ca2+-triggered synaptic vesicle exocytosis and clathrin-mediated endocytosis at a central synapse.

We have tested whether action potential-evoked Ca2+ influx is required to initiate clathrin-mediated synaptic vesicle endocytosis in the lamprey reticulospinal synapse. Exo- and endocytosis were temporally separated by a procedure involving tonic action potential stimulation and subsequent removal of extracellular Ca2+ (Ca2+e). A low concentration of Ca2+ ([Ca2+]e of 11 microM) was found to be required for the induction of early stages of endocytosis. However, the entire endocytic process, from the formation of clathrin-coated membrane invaginations to the generation of synaptic vesicles, proceeded in the absence of action potential-mediated Ca2+ entry. Our results indicate that the membrane of synaptic vesicles newly incorporated in the plasma membrane is a sufficient trigger of clathrin-mediated synaptic vesicle endocytosis.

Phenotypic analysis of cellular immune response in Swiss albino mice immunized with antigen extract from exposed B. alexandrina subjected to UV-, gamma irradiation and praziquantel treatment.

Mice were immunized (2x) subcutaneously at zero and 21st day with different antigen preparations. Splenocytes and mesenteric lymph nodes (MLN) cells were obtained from both immunized and control groups at 14th 21st and 42nd day post the 2nd immunization. The direct immunofluorescent assay was applied using anti mouse CD3+, D4+, CD8+, and IgG monoclonal antibodies to detect panel T-cells, T-cell subsets and B-cells respectively. The observations revealed that the splenic CD3+ T-cells did not show any considerable interrelation either between experimental groups or different time intervals post-immunization (P.I.). Meanwhile, UV-irradiated and PZQ-treated groups showed slight increase at the 2nd and 4th week P.I. as compared with the control group. The UV- and gamma-irradiated groups showed a significant elevation in CD4+ T-cells when compared with control group throughout the time of experiment, whereas, PZQ-treated group showed a significant increase at the 2nd and 4th week P.I. only. The frequency of CD8+ T-cells was elevated in all the experimental groups as compared with control group at 2nd, 4th and 6th week P.I. The MLN CD3+, CD4+ and CD8+ T-cells did not reveal any considerable changes between the experimental groups and control throughout the time of the experiment. A gradual decrease was observed in all the experimental groups by proceeding the time of the experiment. Splenic and MLN B-cells did not show considerable changes between the experimental groups and control group during the course of the experiment. It is concluded that these antigens could possibly stimulate cellular immune response upon their use as a protective antigens.

Analysis of factors affecting outcome after hepatectomy of patients with liver cirrhosis and small hepatocellular carcinoma.

OBJECTIVE: To review our policy of screening patients at risk of developing hepatocellular carcinoma (HCC), and to present 10 years' experience of hepatic resection for small HCC (< 5 cm). DESIGN: Retrospective study. SETTING: Teaching hospital, Germany. SUBJECTS: 861 patients with hepatic cirrhosis and oesophageal varices of whom 151 (18%) had HCC confirmed histologically; 30 of these tumours (20%) were less than 5 cm in diameter and suitable for resection. INTERVENTIONS: Segmentectomy (n = 14) bisegmentectomy (n = 10), and oncologically defined wedge resection (n = 6). MAIN OUTCOME MEASURES: Mortality, morbidity, and survival. RESULTS: 4 patients died within 30 days of liver failure and sepsis (n = 20, liver failure (n = 1), and bronchopneumonia (n = 1). The main beneficial prognostic factors were Child classification, donation of autologous blood, and an encapsulated tumour. The main indicators of a poor prognosis were invasion of the liver, venous invasion, invasion of the resection margin, and the presence of microsatellite tumours and nodules. 12 of the 26 survivors developed recurrences during the first five years postoperatively (46%). Kaplan-Meier survival curves showed that survival at 1 year was 80%, at 3 years 65%, and 5 years 50%, and at 10 years 30%. CONCLUSION: Hepatic resection is a useful treatment for small HCC, but its success depends on early detection and careful selection of patients.

Identification of surface tegumental antigens of normal and irradiated schistosomula.

Sodium dodecyl sulphate- poly acrylamide gel electronphoresis (SDS-PAGE) fractionation of tegumental surface antigens (STEG-Ags.) of 7-day cultured normal and irradiated schistosomula showed no obvious qualitative differences. The observed polypeptide bands of both normal irradiated STEG-Ags. were almost identical and have similar corresponding molecular weights. The immunoblotting assay, using different types of mouse sera, revealed similarity between the bands of both normal and irradiated STEG-Ags. recognized by each type of mouse serum. No qualitative rather than quantitative differences have been observed. The quantitative differences were reflected in intensively staining of some bands from normal STEG-Ag. rather than their corresponding bands of the same molecular weights from irradiated STEG-Ag.